Algeria
Algeria
Chlef, Algeria
Chlef, Algeria
Chlef, Algeria
Chlef, Algeria
Chlef, Algeria
Chlef, Algeria
Rhamnus alaternus L. is a Rhamnaceae shrub and a popular traditional medicine in Algeria. The present research objective was to investigate the antioxidant, genotoxic, and antigenotoxic properties of R. alaternus methanolic leaf extract. Antiradical scavenging activity was tested by α, α-diphenyl-β-picrylhydrazyl free radical scavenging and β-carotene bleaching method. DNA damage and repair were measured by the Allium cepa test with sodium azide as a mutagenic agent. Mitotic index and chromosomal aberrations were calculated by microscopy of meristem roots stained with 2% carmine acetic. The methanolic extract of R. alaternus leaves inhibited the free radical DPPH (IC50 = 0.74 ± 0.30 mg/mL) and prevented the oxidation of β-carotene (50.71 ± 4.17%). The root phenotyping showed that sodium azide changed their color and shape, decreased their stiffness, and significantly reduced their length. The roots treated with both R. alaternus leaf extract and sodium azide demonstrated a better root growth. The roots treated with the methanolic extract were much longer than the control roots (P < 0.001). The microscopy images of root meristem treated with the sodium azide mitodepressant agent showed significant chromosomal aberrations, which indicated a disruption of the cell cycle. The R. alaternus leaf extract appeared to have a beneficial effect on cytotoxicity. The antioxidant properties of R. alaternus L. makes this plant an excellent genoportector.
Rhamnus alaternus L., antioxidant activity, Allium cepa, chromosomal aberrations, antigenotoxicity, mitotic index
INTRODUCTION
Radical oxygen species lead to cell damage, which
can induce genetic instability responsible for many
pathological processes. This damage can be repaired
by some natural compounds, e.g. radical scavengers
and powerful protective antioxidants [1]. Kitagishi et al.
proved that medicinal herbs could one day become
a promising therapeutic means of cancer therapy [2].
According to Dayani et al., antioxidant, antiinflammatory,
and anti-apoptotic properties of plants
and their derivatives make them good radioprotectors
against the mutagenic action of X-rays [3].
Phytotherapy relies on medicinal plants and their
active compounds. Rhamnus alaternus L. (Rhamnaceae
family), also called imlilesse or safir in the North of
Algeria, is well known for its biological properties [4].
Zeouk and Bakheti reported that a decoction of the
aerial part of the R. alatrenus leaves and branches has
been widely used in traditional medicine to lower blood
pressure and treat hepatitis, icterus, musculoskeletal
disorders, and gastrointestinal diseases. They also serve
as a cataplasm for skin infections [5].
Previous findings proved that R. alaternus extracts
possess potential antioxidant, cytotoxic antimutagenic,
antigenotoxic, and antimicrobial activities [5–8]. In
their bibliographic review, Nekkaa et al. focused on
the phytochemical and pharmacological properties of
R. alaternus [4]. Its leaf extracts were rich in flavonoids,
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tannins, and anthocyanins, which explains their
potential antigenotoxic and antimutagenic activity.
Bhouri et al. isolated kaempferol 3-O-b-isorhamninoside
and rhamnocitrin 3-O-b-isor-hamninoside
from R. alternus leaves [9]. These flavonoids
are effective free radical scavengers and potent
antigenotoxics. However, they can induce apoptosis in
human lymphoblastoid cells by the extrinsic apoptotic
mechanism including DNA fragmentation, PARP
cleavage, and active caspase-3 and caspase-8 [10].
Oligomer flavonoid extract from R. alaternus leaves
proved to have a good potential for alternative
antimelanoma therapies [11].
Although some plant remedies have welldocumented
protective effects and alleviate many
diseases, cytotoxicity studies are very important for
developing new drugs. Gadouche et al. described the
toxic effect of Aristolochia longa L. and Calycotome
spinosa L. on the blood cells and concluded that
it should be studied on cancer cells [12]. Natural
antioxidants can even protect human organism against
the cytotoxic and mutagenic effects of xenobiotics.
In this research, we analyzed the genotoxic and
DNA damage protecting activity of R. alaternus
leaf extract by using the Allium cepa assay with azide
sodium as a mutagen agent.
STUDY OBJECTS AND METHODS
Plant material. The research featured Rhamnus
alaternus eu-alaternus L., a subspecies of Rhamnus
alaternus L. The samples were collected in the Bissa
forest located in the north of the Chlef province
(Algeria). This species of Algerian flora was identified
by Dr. Belhacine, a botanist from the Chlef University
[13].
The R. alaternus leaves were dried in the dark for 10
days. After that, they were ground into a fine powder
and kept in an airtight container, and 10 g of the dry
powder was macerated in 100 mL of petroleum ether for
24 h with stirring. The mix was filtered on Whatman No.
1 paper. The maceration included 100 mL of methanol.
After filtration, the marc was evaporated in a rotary
evaporator at 39°C. The extract obtained was stored at
4°C until use [14].
Quantitative analysis and antioxidant
activity. The polyphenols were assayed according
to the method developed by Raafat and Samy
[15]. The amount of total polyphenols was
determined spectrophotometrically using the Folin-
Ciocalteu reagent and deduced from a calibration
curve established with gallic acid (0–1 mg/mL).
The results were expressed in mg of gallic acid
equivalent per g of dry matter (mg GAE/g of dry matter).
The mix included 250 μL of Folin Ciocalteu’s phenol
reagent, 50 μL of each concentration prepared from
stock solution, and 500 μL of 20% Na2CO3 aqueous
solution. After vortexing, the solution was adjusted
with 5 mL of distilled water. After 30 min of incubation,
the absorbance was measured at 765 nm. The same
procedure was carried out with the extract obtained
from R. alaternus leaves.
The flavonoid content was assayed according to the
method developed by Hmid et al. [16]. After 1 mL of
extract was added to 1 mL of 2% aluminum chloride,
the absorbance was determined at 430 nm after 10 min
of incubation. Quercetin served as calibration curve
standard and was established from the concentration of
40 μg/mL of stock solution. Total flavonoids content in
the extract was expressed as mg quercetin equivalents
per g of sample (mg EQ/g of dry matter).
The DPPH assay followed the method described by
Burits and Bucar [17]. The R. alaternus extract had the
following concentrations: 0.2, 0.4, 1.0, and 2.0 mg/mL.
We mixed 50 μL of each concentration with 5 mL of
0.004% DPPH. The absorbance was taken at 517 nm
after 30 min of incubation. The results were compared
to ascorbic acid, which was used as standard antioxidant
and handled under the same conditions. The percentage
of inhibition and IC50 were calculated according to
Sharififar et al. [18]. The percentage inhibition was
calculated using the following equation:
IC50 is the concentration of extract required for 50%
inhibition of DPPH. It was calculated using a linear
regression analysis.
The β-carotene-linoleic acid assay was performed
according to the method described by Kartal et al. [19].
The emulsion included 0.5 mg of β-carotene, 1 mL
of chloroform, 25 μL of linoleic acid, and 200 mg of
tween 40. The chloroform was eliminated in a rotary
evaporator under vacuum, and 100 mL of distilled
oxygen-saturated water was added to the emulsion.
Subsequently, 350 μL of the extract at a concentration
of 2 mg/mL was mixed with 2.5 mL of the emulsion.
After 48 h of incubation, the absorbance was registered
at 490 nm and compared with that obtained with
butylohydroxytoluene (BHT), which served as a
standard antioxidant and was prepared under the same
conditions. The inhibition percentage of bleaching
(I, %) was measured for each assay using the following
equation:
Allium cepa assay. The Allium cepa assay was
performed according to Tedesco and Laughinghouse with
some modifications [20]. The onion bulbs were kept in
a culture medium that included 60 mg/L of CaSO4, 60
mg/L of MgSO4, 96 mg/L of NaHCO3, and 4 mg/L of
KCl. They were incubated at 25°C for 72 h until the
roots reached 2 cm. Seven onion bulbs were utilized for
each treatment as follows:
Sample 1: culture medium + distilled water;
Sample 2: culture medium + sodium azide
(50 mg/mL);
Sample 3: culture medium + sodium azide (100 mg/mL);
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Sample 4: culture medium + methanolic extract
(50 mg/mL);
Sample 5: culture medium + methanolic extract
(100 mg/mL);
Sample 6: culture medium + sodium azide
(100 mg/mL) + methanolic extract (50 mg/mL);
Sample 7: culture medium + sodium azide
(100 mg/mL) + methanolic extract (100 mg/mL).
The effect of the different treatments on the growth
(cm) of the A. cepa roots was measured at different
time intervals: 0, 24, 48, and 72 h. In parallel, the roots
were tested for color, shape, and stiffness. After each
time interval, the roots were collected for microscopic
observation of the meristem cells and stored in 70%
ethanol for later use. The roots were fixed in acetic acid
and ethanol solution (1:3) for 24 h. After triple rinsing
with distilled water, the roots were hydrolyzed with
HCl (1N) and incubated in a hot water bath at 60°C for
10 min. After the hydrolysis, the roots were rinsed
once again in distilled water and stained with 2%
acetic carmine in a hot water bath at 60°C for 10 min.
After incubation, the terminal meristem cells of the
colored roots were cut with a scalpel under a binocular
magnifier. The meristem regions were crushed
manually between blade and coverslip to visualize the
chromosomes and the different stages of cell division.
Meristem cells were counted for each sample and
tested for normal or abnormal cell division in search for
mutations. The mitotic index and the rate of aberrant
cells of each bulb were calculated by the following
formula [21]:
Statistical analysis. The experimental data were
analyzed using the ExcelSTAT software. The research
also included the ANOVA variance analysis, followed by
the Tukey’s test. The statistically highly significant value
was P < 0.001.
RESULTS AND DISCUSSION
The total phenol content in Rhamnus alaternus L.
leaves was 32.6 ± 1.82 mg GAE/g DM, and the total
flavonoid content was 27.58 ± 0.01 mg EQ/g DM. The
methanolic extract of R. alaternus leaves demonstrated
a moderate efficiency against free radicals emitted
by linoleic acid (50.71 ± 4.17%). Its capacity to beat
free radical of DPPH (I% = 80.39 ± 2.33%, IC50 =
0.74 ± 0.30 mg/mL) was close to that of ascorbic acid, i.e.
96.80 ± 9.98% with IC50 = 0.37 ± 1.1 mg/mL (Table 1).
Plants are excellent indicators of the cytotoxic,
cytogenetic, and mutagenic effects of environmental
chemicals. They can serve as an alternative for detecting
possible genetic damage in cells [22]. Genotoxicity
studies were carried out by the Allium cepa assay. This
method provides a convenient in vivo model to evaluate
cell cycle alterations induced by mutagens [20].
The A. cepa roots treated with distilled water had no
morphological change: the growth rate was good, the
color was whitish, and the roots were rigid and bulky.
However, the roots treated with sodium azide (50 and
100 mg/mL) changed the color and shape of the roots, as
well as reduced their rigidity (+very brittle) and growth
rate.
The methanolic leaf extract of R. alaternus had
no negative effect on the morphology. The samples
demonstrated good growth, strong rigidity, and whitish
color. Their morphology was comparable to the control
roots. The roots incubated in both methanolic extract
and sodium azide had a phenotype close to the control
roots. They were better preserved than the roots treated
only with sodium azide. The roots of this sample showed
good growth, and the color was comparable to that of the
control roots (Fig. 1).
Table 2 shows a highly significant decrease in the
growth of the A. cepa roots treated with sodium azide
at two concentrations (50 and 100 mg/mL) at three time
intervals. The data obtained from the sample treated
with 50 mg/mL of sodium azide after 48 h was found
insignificant (P < 0.001).
The roots treated with the methanolic extract of
R. alaternus leaves showed highly significant growth
(P < 0.001) after 24 and 48 h. The roots reached 8 cm
after 72 h (P < 0.001) and were longer than those treated
with distilled water (7 cm).
The difference in length for the antigenotoxicity
test was highly significant after 48 and 72 h and
not significant after 24 h. The roots demonstrated a
clearly significant improvement in the diameter after
Parameter Leaves of R. alaternus Ascorbic acid Butylated
hydroxytoluene
Polyphenol, mg GAE/g dry matter
Flavonoids, mg QE/g dry matter
DPPH, % (R. alaternus extract concentration
= 1 mg/mL)
IC50, mg/mL
β-carotene bleaching, % (R. alaternus extract
concentration = 2 mg/mL)
32.60 ± 1.82
27.58 ± 0.01
80.39 ± 2.33
0.74 ± 0.30
50.71 ± 4.17
––
96.80 ± 9.98
0.37 ± 1.10
–
–––
–
98.84 ± 1.69
Table 1 Total phenolic content and total flavonoid content of Rhamnus alaternus leaves, DPPH inhibition, IC50, and % bleaching
of β-carotene
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Figure 1 Morphological aspects of Allium cepa roots: (a) control group; (b) sodium azide (50 mg/mL); (c) sodium azide
(100 mg/mL); (d) methanolic extract (50 mg/mL); (e) methanolic extract (100 mg/mL); (f) sodium azide (100 mg/mL) + methanolic
extract (50 mg/mL); (g) sodium azide (100 mg/mL) + methanolic extract (100 mg/mL)
a b c d e f g
72 h. It was 0.26 and 0.13 cm respectively for the two
extract concentrations.
Microscopy revealed no abnormalities or
disturbances in mitotic division: chromosome integrity
maintained its high mitotic index (69.76 ± 7.01%),
and no chromosomal aberrations were registered
(Fig. 2, Table 2).
The microscopy of the roots stained with 2% acetic
carmine after treatment with two concentrations of
sodium azide revealed several chromosomal anomalies
with disruption of all the stages of cell division
(Figs. 3 and 4, Table 2). Several cells contained
C-mitosis, S-mitosis, chromosomal breaks, bridges,
and uneven distribution of chromosomes, which led to
disturbed anaphases, metaphases, and telophases. These
anomalies were caused by both concentrations of azide;
however, they were much more severe at 100 mg/mL of
sodium azide.
Table 2 ΔL – differences in length of the Allium cepa roots before and after each treatment, % mitotic index, and chromosomal
aberrations
Treatment Time, h ΔL, cm Mitotic index, % Chromosomal
aberrations, %
Control 0
24
48
72
2.66 ± 0.56
0.57 ± 0.05
0.84 ± 0.09
0.70 ± 0.24 69.76 ± 7.01
0
Sodium azide (50 mg/mL) 0
24
48
72
2.36 ± 0.48
0.50 ± 0.11**
0.94 ± 0.10
–0.54 ± 0.31**
34.48 ± 10.50
5.03 ± 1.51**
Sodium azide (100 mg/mL) 0
24
48
72
2.84 ± 0.47
–1.12 ± 0.18**
–0.40 ± 0.16**
–0.03 ± 0.12** 29.25 ± 8.50
7.84 ± 2.41**
Methanolic extract (50 mg/mL) 0
24
48
72
2.93 ± 0.19
1.09 ± 0.21**
0.90 ± 0.07**
0.46 ± 0.15 69.54 ± 14.5
0.43 ± 0.53
Methanolic extract (100 mg/mL) 0
24
48
72
2.80 ± 0.47
1.87 ± 0.04**
1.37 ± 0.09**
1.05 ± 0.02 73.66 ± 9.41 0.29 ± 0.49
Sodium azide (100 mg/mL) + methanolic
extract (50 mg/mL)
0
24
48
72
2.71 ± 0.38
0.11 ± 0.05
–0.33 ± 0.12**
0.26 ± 0.18** 51.77 ± 14.44 2.55 ± 1.98
Sodium azide (100 mg/mL) + methanolic
extract (100 mg/mL)
0
24
48
72
2.47 ± 0.57
0.53 ± 0.08
–0.10 ± 0.07**
0.13 ± 0.09**
52.34 ± 8.12
3.34 ± 2.82
ΔL – mean difference in length of Allium cepa roots before and after treatment
**P ≤ 0.001
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Figure 3 Various anomalies caused by sodium azide at 50 mg/mL (100×): (a) binucleated cell; (b) disturbed telophase; (c) disturbed
anaphase; (d) normal anaphase; (e), (f) disturbed metaphase
Figure 4 Chromosomes of Allium cepa roots treated with 100 mg/mL of sodium azide (100×): (a) binucleated cells; (b)
chromosomal break, chromosomal bridge; (c), (e) disrupted (uneven) anaphase; (d) prophase, chromosomal bridge; (f) C-mitosis
Figure 2 Normal mitotic divisions of Allium cepa meristem cells (100×): (a) interphase, prophase, and metaphase; (b) start of
anaphase; (c), (e), (f) anaphase; (d) telophase
a b c d
e f
a b c d
e f
a b c d
e f
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Chromosomal aberrations increased together with
the concentration of sodium azide. The genotoxic effect
was most severe at 100 mg/mL. Both concentrations of
sodium azide reduced the mitotic index, which meant
that sodium azide blocked cell division. On the other
hand, the number of chromosomal aberrations grew
together with sodium azide concentration. They were
represented mainly by C-mitosis, chromosomal bridges
and breaks, and nuclear lesions of binucleate types.
Therefore, sodium azide was an aberration inducer
(Figs. 3 and 4, Table 2).
Sodium azide produced a cytotoxic effect
which led to poor growth and length narrowing. Its
mitodepressive effect decreased mitotic activity
and increased chromosomic abnormality incidence.
Indeed, chemical agents are recognized as factors
involved in the structural and numerical modifications
of chromosomes. As a result, they cause defects in
chromosome segregation, abnormal DNA replication,
and DNA breaks. These chromosomal aberrations
result from clastogenic and aneugenic effects [23]. This
study confirmed the genotoxic effect of sodium azide.
According to Al-Qurainy et al., sodium azide is a
mutagenic metabolite that damages DNA by substituting
one base pair with another [24]. Indeed, the shorter
length of A. cepa roots treated with sodium azide
could be explained by the mitodepressive effect caused
by the apoptosis of meristem cells. Other samples
demonstrated evolution of the normal length, probably,
due to the resumption of mitosis.
Sodium azide induced the development of
chromosome bridges in the meristem cells of
A. cepa roots. According to Neelamkavil and Thoppil,
the chromosomal aberrations and nuclear lesions in
A. cepa root meristems treated with bleaching powder
indicated a genotoxic effect, which confirms that
sodium azide is genotoxic [25]. The clastogenic effects
suggest that bleaching powder caused chromosome and
chromatin breaks, which, in return, led to abnormal
chromosome number, stickiness, breakage, and reunion
of chromosome, as well as to bridges during mitotic
division [26, 27].
The mitotic index was higher in the roots treated
with two concentrations of the R. alaternus extract
than in those treated with distilled water. Therefore,
the extract induced cell division and, subsequently,
produced a genoprotective effect. Moreover, the
number of cells in division was high with traces
chromosomal aberrations also proven by a marked
root length. The samples treated with 50 mg/mL of
R. alaternus methanolic extract had pycnotic nuclei
and chromosomal breaks (Figs. 5 and 6, Table 2). This
finding confirms the conclusion made by Ben Ammar
et al., who experimented with methanolic, petroleum
ether, chloroform, and aqueous extracts of R. alaternus
leaves and registered no mutagenicity, which means that
R. alaternus is a promising antimutagenic [28].
The antigentoxic effect showed that the mitotic
index was close to that of the control. It had a moderate
chromosomal aberration percentage, chromosomal
bridges and breaks, and a lower C-mitosis (Figs. 7
and 8, Table 2).
A quantitative analysis of the R. alaternus methanolic
extract revealed a lot of polyphenols and flavonoids
and thus a prominent antioxidant effect. This antioxidant
effect might be the cause of the continuous cell division,
mitoprotective activity, and a good DNA protection.
Perron et al. tested 12 polyphenolic compounds,
which demonstrated a 100% ability to inhibit DNA damage
[29]. The polyphenolic compounds had hydroxyl
radicals in their chemical structures, which prevented
oxidative DNA damage.
On the other hand, Silva et al. showed that flavonoids
have special DNA repair mechanisms that enable
them to reduce and repair DNA strand breaks induced
by oxidative stress [30]. Therefore, polyphenols are
effective protectors against oxidative DNA damage.
Figure 5 Chromosomes of Allium cepa roots treated with 50 mg/mL of Rhamnus alaternus leaf extract (100×): (a) prophase; (b) end
of interphase; (c) telophase; (d) pycnotic nucleus; (e) metaphase; (f) anaphase
a b c d
e f
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Figure 6 Chromosomes of Allium cepa roots treated with 100 mg/mL of Rhamnus alaternus leaf extract (100×): (a), (b), (f)
telophase/cytodiuresis; (c) anaphase, prophase, and metaphase; (d) anaphase with chromosome breaks; (e) chromosome bridge with
an isolated chromosome
a b c d
e f
Figure 7 Chromosomes of Allium cepa roots treated with 50 mg/mL of methanolic extract and 100 mg/mL of sodium azide (100×):
(a) several prophases; (b) several prophases; (c), (f) metaphase; (d) anaphase; (e) telophase start of prophase
a b c d
e f
Figure 8 Chromosomes of Allium cepa roots treated with 50 mg/mL of methanolic extract and 100 mg/mL of sodium azide (100×):
(a) binucleated; (b) metaphase, anaphase; (c) several prophases; (d) prophase; (e) prophase, metaphase, and anaphase.
a b c d
e f
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CONCLUSION
Medicinal plants contain a lot of secondary
metabolites with beneficial therapeutic and pharmacological
properties, which deserve extensive research.
Rhamnus alaternus L. proved to be an effective antioxidant
and mitoprotector that can boost the development
of pharmacognosy and produce new herbal drugs
for the pharmaceutical industry. The genoportective
effect of R. alaternus leaf extract could be a source of
new cancer drugs and protect human genome from the
side effects of chemical treatment.
CONTRIBUTION
L. Gadouche conceived and designed the analysis,
performed the biological experiments, and wrote the
paper. A. Zidane and K. Zerrouki contributed to the data
analysis and revised the paper. A. Ababou performed
the statistical analysis. I. Bachir Elazaar, D. Merabet,
W. Henniche, and S. Ikhlel performed the biological
experiments. All the authors revised the manuscript for
publication.
CONFLICT OF INTEREST
The authors declare that there was no potential
conflict of interests regarding the publication of this
article.
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